Ultrasound microbubble potentiated enhancement of hyperthermia-effect in tumours.

It is now well established that for tumour growth and survival, tumour vasculature is an important element. Studies have demonstrated that ultrasound-stimulated microbubble (USMB) treatment causes extensive endothelial cell death leading to tumour vascular disruption. The subsequent rapid vascular collapse translates to overall increases in tumour response to various therapies. In this study, we explored USMB involvement in the enhancement of hyperthermia (HT) treatment effects. Human prostate tumour (PC3) xenografts were grown in mice and were treated with USMB, HT, or with a combination of the two treatments. Treatment parameters consisted of ultrasound pressures of 0 to 740 kPa, the use of perfluorocarbon-filled microbubbles administered intravenously, and an HT temperature of 43°C delivered for various times (0-50 minutes). Single and multiple repeated treatments were evaluated. Tumour response was monitored 24 hours after treatments and tumour growth was monitored for up to over 30 days for a single treatment and 4 weeks for multiple treatments. Tumours exposed to USMB combined with HT exhibited enhanced cell death (p<0.05) and decreased vasculature (p<0.05) compared to untreated tumours or those treated with either USMB alone or HT alone within 24 hours. Deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and cluster of differentiation 31 (CD31) staining were used to assess cell death and vascular content, respectively. Further, tumours receiving a single combined USMB and HT treatment exhibited decreased tumour volumes (p<0.05) compared to those receiving either treatment alone when monitored over the duration of 30 days. Additionally, tumour response monitored weekly up to 4 weeks demonstrated a reduced vascular index and tumour volume, increased fibrosis and lesser number of proliferating cells with combined treatment of USMB and HT. Thus in this study, we characterize a novel therapeutic approach that combines USMB with HT to enhance treatment responses in a prostate cancer xenograft model in vivo.

Role of Acid Sphingomyelinase and Ceramide in Mechano-Acoustic Enhancement of Tumor Radiation Responses.

Background: High-dose radiotherapy (>8-10 Gy) causes rapid endothelial cell death via acid sphingomyelinase (ASMase)-induced ceramide production, resulting in biologically significant enhancement of tumor responses. To further augment or solicit similar effects at low radiation doses, we used genetic and chemical approaches to evaluate mechano-acoustic activation of the ASMase-ceramide pathway by ultrasound-stimulated microbubbles (USMB). Methods: Experiments were carried out in wild-type and acid sphingomyelinase (asmase) knockout mice implanted with fibrosarcoma xenografts. A cohort of wild-type mice received the ASMase-ceramide pathway inhibitor sphingosine-1-phosphate (S1P). Mice were treated with varying radiation doses, with or without a priori USMB exposure at different microbubble concentrations. Treatment response was assessed with quantitative 3D Doppler ultrasound and immunohistochemistry at baseline, and at three, 24, and 72 hours after treatment, with three to five mice per treatment group at each time point. All statistical tests were two-sided. Results: Results confirmed an interaction between USMB and ionizing radiation at 24 hours (P < .001), with a decrease in tumor perfusion of up to 46.5% by three hours following radiation and USMB. This peaked at 24 hours, persisting for up to 72 hours, and was accompanied by extensive tumor cell death. In contrast, statistically nonsignificant and minimal tumor responses were noted in S1P-treated and asmase knockout mice for all treatments. Conclusions: This work is the first to confirm the involvement of the ASMase-ceramide pathway in mechanotransductive vascular targeting using USMB. Results also confirm that an acute vascular effect is driving this form of enhanced radiation response, and that it can be elicited at low radiation doses (<8-10 Gy) by a priori USMB exposure.

Tumour Vascular Shutdown and Cell Death Following Ultrasound-Microbubble Enhanced Radiation Therapy.

High-dose radiotherapy effects are regulated by acute tumour endothelial cell death followed by rapid tumour cell death instead of canonical DNA break damage. Pre-treatment with ultrasound-stimulated microbubbles (USMB) has enabled higher-dose radiation effects with conventional radiation doses. This study aimed to confirm acute and longitudinal relationships between vascular shutdown and tumour cell death following radiation and USMB in a wild type murine fibrosarcoma model using in vivo imaging. Methods: Tumour xenografts were treated with single radiation doses of 2 or 8 Gy alone, or in combination with low-/high-concentration USMB. Vascular changes and tumour cell death were evaluated at 3, 24 and 72 h following therapy, using high-frequency 3D power Doppler and quantitative ultrasound spectroscopy (QUS) methods, respectively. Staining using in situ end labelling (ISEL) and cluster of differentiation 31 (CD31) of tumour sections were used to assess cell death and vascular distributions, respectively, as gold standard histological methods. Results: Results indicated a decrease in the power Doppler signal of up to 50%, and an increase of more than 5 dBr in cell-death linked QUS parameters at 24 h for tumours treated with combined USMB and radiotherapy. Power Doppler and quantitative ultrasound results were significantly correlated with CD31 and ISEL staining results (p < 0.05), respectively. Moreover, a relationship was found between ultrasound power Doppler and QUS results, as well as between micro-vascular densities (CD31) and the percentage of cell death (ISEL) (R2 0.5-0.9). Conclusions: This study demonstrated, for the first time, the link between acute vascular shutdown and acute tumour cell death using in vivo longitudinal imaging, contributing to the development of theoretical models that incorporate vascular effects in radiation therapy. Overall, this study paves the way for theranostic use of ultrasound in radiation oncology as a diagnostic modality to characterize vascular and tumour response effects simultaneously, as well as a therapeutic modality to complement radiation therapy.

Microbubble-based enhancement of radiation effect: Role of cell membrane ceramide metabolism.

Ultrasound (US) stimulated microbubbles (MB) is a new treatment approach that sensitizes cancer cells to radiation (XRT). The molecular pathways in this response remain unelucidated, however, previous data has supported a role for cell membrane-metabolism related pathways including an up regulation of UDP glycosyltransferase 8 (UGT8), which catalyzes the transfer of galactose to ceramide, a lipid that is associated with the induction of apoptotic signalling. In this study, the role of UGT8 in responses of prostate tumours to ultrasound-stimulated microbubble radiation enhancement therapy is investigated. Experiments were carried out with cells in vitro and tumours in vivo in which UGT8 levels had been up regulated or down regulated. Genetically modified PC3 cells were treated with XRT, US+MB, or a combination of XRT+US+MB. An increase in the immunolabelling of ceramide was observed in cells where UGT8 was down-regulated as opposed to cells where UGT8 was either not regulated or was up-regulated. Clonogenic assays have revealed a decreased level of cellular survival with the down-regulation of UGT8. Xenograft tumours generated from stably transfected PC3 cells were also treated with US+MB, XRT or US+MB+XRT. Histology demonstrated more cellular damage in tumours with down-regulated UGT8 in comparison with control tumours. In contrast, tumours with up-regulated UGT8 had less damage than control tumours. Power Doppler imaging indicated a reduction in the vascular index with UGT8 down-regulation and photoacoustic imaging revealed a reduction in oxygen saturation. This was contrary to when UGT8 was up regulated. The down regulation of UGT8 led to the accumulation of ceramide resulting in more cell death signalling and therefore, a greater enhancement of radiation effect when vascular disruption takes place through the use of ultrasound-stimulated microbubbles.

Assessment of tumor response to radiation and vascular targeting therapy in mice using quantitative ultrasound spectroscopy.

PURPOSE: It is now recognized that the tumor vasculature is in part responsible for regulating tumor responses to radiation therapy. However, the extent to which radiation-based vascular damage contributes to tumor cell death remains unknown. In this work, quantitative ultrasound spectroscopy (QUS) methods were used to investigate the acute responses of tumors to radiation-based vascular treatments. METHODS: Tumor xenografts (MDA-MB-231) were treated with single radiation doses of 2 or 8 Gy alone, or in combination with pharmacological agents that modulate vascular radiosensitivity. The midband fit, the slope, and the 0-MHz intercept QUS parameters were obtained from a linear-regression fit to the averaged power spectrum of frequency-dependent ultrasound backscatter and were used to quantify acute tumor responses following treatment administration. Power spectrums were extracted from raw volumetric radio-frequency ultrasound data obtained before and 24 h following treatment administration. These parameters have previously been correlated to tumor cell death. Staining using in situ end labeling, carbonic anhydrase 9 and cluster of differentiation 31 of tumor sections were used to assess cell death, oxygenation, and vasculature distributions, respectively. RESULTS: Results indicate a significant midband fit QUS parameter increases of 3.2 ± 0.3 dBr and 5.4 ± 0.5 dBr for tumors treated with 2 and 8 Gy radiation combined with the antiangiogenic agent Sunitinib, respectively. In contrast, tumors treated with radiation alone demonstrated a significant midband fit increase of 4.4 ± 0.3 dBr at 8 Gy only. Preadministration of basic fibroblast growth factor, an endothelial radioprotector, acted to minimize tumor response following single large doses of radiation. Immunohistochemical analysis was in general agreement with QUS findings; an R(2) of 0.9 was observed when quantified cell death was correlated with changes in midband fit. CONCLUSIONS: Results from QUS analysis presented in this study confirm that acute tumor response is linked to a vascular effect following high doses of radiation therapy. Overall, this is in agreement with previous reports suggesting that acute tumor radiation response is regulated by a vascular-driven response. Data also suggest that Sunitinib may enhance tumor radiosensitivity through a vascular remodeling process, and that QUS may be sensitive to changes in tissue properties associated with vascular remodeling. Finally, the work also demonstrates the ability of QUS methods to monitor response to radiation-based vascular strategies.